Proline hydroxylase and uses thereof

ABSTRACT

Provided are a proline hydroxylase and uses thereof. The proline hydroxylase comprises having the amino acid sequence of SEQ ID NO: 2 with the exception of a mutation of one or more amino acids; wherein the mutation of one or more amino acids must comprises E27K, and the mutation of one or more amino acids selected from the group consisting of: H14R, L16N, T25R, F26L, E27K, D30S, S33N, E34N, E34G, E34L, E34S, E34D, Y35W, Y35K, S37W, S37F, S37E, S37N, S37T, S37C, W40F, K41E, D54G, H55Q, S57L, I58T, I58Y, I58A, I58R, I58V, I58S, I58C, K86P, T91A, F95Y, C97Y, I98V, K106V, K106T, K106Q, F111S, K112E, K112R, S154A, K162E, L166M, I118F, I118V, I118R, H119R, H119F, I120V, K123D, K123N, K123Q, K123S, K123I, K123T, T130N, D134G, V135K, N165H, D173G, K209R, I223V and S225A, and having proline hydroxylase activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/344,779 filed 24 Apr. 2019 entitled “PROLINE HYDROXYLASE AND USESTHEREOF,” which issued Mar. 30, 2021 as U.S. Pat. No. 10,961,516 B2,which is a national phase filing of Patent Cooperation Treatyapplication no. PCT/CN2016/104670 filed 4 Nov. 2016 entitled “PROLINEHYDROXYLASE AND USES THEREOF,” which are each hereby incorporated hereinby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII file was created 23 Apr. 2019, isnamed “F20201210_P281926WO-US02_DIV_SequenceList_PN144419KLY.txt”, is533 bytes in size, was filed in parent U.S. patent application Ser. No.16/344,779 filed 24 Apr. 2019, and contains a sequence listing identicalto the sequence listing filed in the corresponding internationalapplication no. PCT/CN2016/104670 created and filed on 4 Nov. 2016.

TECHNICAL FIELD

The application relates to genetic engineering and enzyme engineering,and in particular, to a proline hydroxylase and uses thereof.

BACKGROUND

A derivative of proline is an important structure unit of pharmaceuticalsynthesis, especially hydroxyproline which belongs to rare amino acid inthe nature world, and is a starting raw material for synthesis of manyimportant pharmaceuticals. According to different proline hydroxylationpositions and space structures, there are eight isomers of thehydroxyproline totally, herein 3-hydroxy-L-proline and4-hydroxy-L-proline are used as the important raw materials of multiplepharmaceuticals of antibiotics, enzyme inhibitors, antineoplastics,antihypertensive agents and new-type stomach medicines and the like, andare a hot point of existing biosynthesis research. In addition, thehydroxyproline may be a source of a natural product or chemicalsynthesis, for example, the hydroxyproline may be a plant material or ahydrolysate of collagen, or use allyl bromide, diethylacetaminopropionic acid, D-glutamic acid and Beta-alanine as thestarting raw materials for performing the chemical synthesis.

Similarly, the derivative L-hydroxypiperidine acid of the hydroxyprolineis also the important structure unit of the pharmaceutical synthesis,and is an important intermediate of the synthesis of a Beta-lactamaseinhibitor and a TNF-A invertase inhibitor. The L-hydroxypiperidine maybe prepared through separation of plants or other natural materials orthrough a chemical synthesis method similarly. But a complicatedseparating and purifying method or a complicated synthesis process isneeded, and it is difficult to realize large-scale industrialproduction. A mode of performing hydroxylation on piperidine acidthrough hydroxylase is an ideal method for acquiring thehydroxypiperidine acid. Enzyme frequently applied at present is theproline hydroxylase, it is a type of ketoglutarate-dependentdioxygenase, and A-oxoglutarate and 02 are needed as a common substrate,and iron ions are used as a cofactor.

Many microorganisms contain the proline hydroxylase, for example,Sinorhizobium meliloti, Streptomyces sp. strain THI, and Glarealozoyensis. Three types of frequently-usedcis-form-proline-3-hydroxylase, cis-form-proline-4-hydroxylase andanti-form-proline-4-hydroxylase may respectively catalyze L-proline togenerate cis-form-3-hydroxy-L-proline, cis-form-4-hydroxy-L-proline andanti-form-4-hydroxy-L-proline. But in a catalytic reaction of theproline hydroxylase to the proline and the proline derivative, someinevitable problems are existent, for example, a conversion rate is low,and a position isomer is generated by catalysis, the large-scaleindustrial production may not be realized. After thecis-form-proline-4-hydroxylase from the Sinorhizobium meliloti isgenetically modified, the conversion rate of L-piperidine acidhydroxylation and specifity of enzyme catalysis are greatly improved,but 10% of the position isomer (2S,3R)-3-hydroxypiperidine-2-carboxylicacid is still generated (W02013169725A2). So, it has importantsignificance to industrial synthesis of the hydroxyproline and thederivative thereof that a type of the enzyme capable of specifically andrapidly catalyzing the hydroxylation of the proline and the prolinederivative is found out.

SUMMARY

A main purpose of the application is to provide a proline hydroxylaseand uses thereof, and solve the problem of poor selectivity of prolinehydroxylation enzyme catalysis in the prior art.

In order to realize the above purpose, according to one aspect of theapplication, a proline hydroxylase is provided, the proline hydroxylasecomprises: (a) a protein having an amino acid sequence as shown in SEQID NO: 2; (b) a protein having an amino acid sequence of SEQ HD NO: 2with a mutation of one or more amino acids and having a prolinehydroxylase activity; or (c) a protein retaining the mutation of one ormore amino acids as in (b), and having the proline hydroxylase activityand having at least 78% homology with the amino acid sequence of theprotein in (b).

Further, a site of the mutation is selected from one or more of thegroup consisting of H14, S16, T25, F26, E27, D30, S33, E34, Y35, S37,I39, W40, K41, D54, H55, S57, I58, K86, T91, F95, C97, 198, K106, F111,K112, K162, L166, I118, H119, I120, K123, T130, D134, V135, S154, N165,D173, K209, I223 and S225.

Further, the mutation comprises any one or more of the group consistingof: H14R, 516N, T25G, T25R, F26L, E27K, D30S, S33N, E34N, E34G, E34L,E34S, E34D, Y35W, Y35K, S37W, S37F, S37E, S37N, S37T, S37C, I39K, 139R,W40F, K41E, D54G, H55Q, S57L, I58T, I58Y, I58A, I58R, I58V, I58S, I58C,K86P, T91A, F95Y, C97Y, I98V, K106V, K106T, K106Q, F111S, K112E, K112R,S154A, K162E, L166M, I118F, I118V, I118R, H119R, H119F, I120V, K123D,K123N, K123Q, K123S, K123I, K123T, T130N, D134G, V135K, N165H, D173G,K209R, I223V, and S225A.

Further, the mutation comprises any one of combinations selected fromthe group consisting of: E27K+Y35W/K, E27K+I39K/R, E27K+K123D/I/Q/S,E27K+N165H, I39K/R+Y35W/K, I39K/R+K123D/I/Q/S, I39K/R+N165H, K123D+W40F,K123D+Y35W/K, E27K+I39K/R+K123D/1/Q/S, K123D/I/Q/S+N 165H,537C/E/F/N/W/T+I223V, E27K+Y35W/K+I39K/R, E27K+537C/E/F/N/W/T+I39K/RE27K+E34N/G/L/D/S+I39K/R, E27K+I39K/R+D30S, E27K+I39K/R+I118F/V/R,E27K+I39K/R+I98V, 537C/E/F/N/W/T+I223V+N165H,Y35W/K+537C/E/F/N/W/T+W40F, 537C/E/F/N/W/T+I223V+K123D/1/Q/S,E27K+I39K+Y35W/K+537C/E/F/N/W/T, E27K+I39K/R+S37C/E/F/N/W/T+K123D/I/Q/S,E27K+I39K/R+K106Q+K112E, E27K+I39K/R+Y35W/K+S37C/E/F/N/W/T+K123D/I/Q/S,E27K+I39K/R+S37C/E/F/N/W/T+I58A/C/R/S/T/V/Y,E27K+S37C/E/F/N/W/T+I223V+K123D/I/Q/S,S37C/E/F/N/W/T+I39K/R+I223V+K123D/I/Q/S,E27K+S37C/E/F/N/W/T+I39K/R+K123D/I/Q/S+I98V,E27K+S37C/E/F/N/W/T+I39K/R+K123D/I/Q/S+I223V,F26L+E27K+I39K/R+K123D/I/Q/S, I223V+S37C/E/F/N/W/T+E27K+I39K/R,I223V+S37C/E/F/N/W/T+E27K+N165H,E27K+S37C/E/F/N/W/T+I39K/R+I98V+K123D/I/Q/S+I223V, K106Q+K112E+I223V,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+K123D/I/Q/S,E27K+I39K/R+K123D/I/Q/S+N165H, H14R+E34G+K106Q+K112E+I223V,T25G/R+E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+K86P,K123D/I/Q/S+Y35W/K+I120V, E27K+D30S+I39K/R+I58A/C/R/S/T/V/Y+K112E,S37C/E/F/N/W/T+I39K/R+N165H,E27K+E34N/G/L/D/S+I39K/R+I58A/C/R/S/T/V/Y+I223V,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+I118F/V/R,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/1/Q/S,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/1/Q/S+I118F/V/R,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D1736+K123D/1/Q/S+N165H,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/+D173G+K123D/I/Q/S,H14R+E27K+D30S+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,T25G/R+E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/1/Q/S+I118F/V/R+N165H, H14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+Y35W/K+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+K123D/I/Q/S+I223V,H14R+E27K+E34N/G/L/D+I39K/R+I58A/C/R/S/T/V/Y+I98V+K106V/T/Q+K112E/R+I223VandH14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I118F/V/R+I223V,wherein, ‘/’ represents ‘or’.

In order to realize the above purpose, according to one aspect of theapplication, a DNA molecule is provided, wherein the DNA moleculeencodes any one of the proline hydroxylase.

In order to realize the above purpose, according to another aspect ofthe application, a recombinant vector is provided, wherein therecombinant vector is connected with the DNA molecule.

Further, the recombinant vector is selected from one of the groupconsisting of: pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+),pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+),pET-20b(+), pET 21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+),pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+),pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+),pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), p-44a(+), pET-49b(+),pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B,pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222,pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18 and pUC-19.

According to another aspect of the application, a host cell is provided,wherein the host cell comprises any one of the recombinant vectors.

Further, the host cell is a prokaryotic cell or a eukaryocyte,preferably the eukaryocyte is a yeast cell.

Further, the host cell is a competent cell, preferably the competentcell is an E. coli BL21 cell or an E. coli W3110 cell.

According to another aspect of the application, a method for producingan L-hydroxyproline derivative is provided, wherein the methodcomprises: using an L-proline derivative as a substrate, and applyingany one of the proline hydroxylases to catalyze hydroxylation of thesubstrate, to obtain the L-hydroxyproline derivative as shown in ageneral formula (I):

wherein R₁ is selected from C₁-C₅ alkylene or C₂-C₅ alkenylene; R₂ isselected form C₀-C₄ alkylene or C₂-C₄ alkenylene; R₃ is selected fromhydroxyl, amino, C₁-C₆ alkoxy, aryloxy, C₁-C₆ alkyl sulfenyl or C₁-C₆aryl sulfenyl; and R₄ is selected from hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl or C₂-C₆ alkynyl.

Further, the method comprises: using α-oxoglutarate and O₂ as a commonsubstrate, using iron ions as a cofactor, applying the prolinehydroxylase to catalyze hydroxylation of the substrate, to obtain theL-hydroxyproline derivative.

Further, the L-hydroxyproline derivative is cis-4-hydroxy-L-proline or(2S,5S)-5-hydroxypiperidine-2-carboxylic acid.

Further, the proline hydroxylase catalyzes hydroxylation of thesubstrate in a temperature of 5˜45 DEG C., preferably 5˜15 DEG C., toobtain the L-hydroxyproline derivative as shown in the general formula(I).

By means of the technical solutions of the present application, byselecting SEQ ID NO: 2 as a base sequence, a mutant containing single ormultiple amino acid residues modified through genetic engineering, or byaltering other amino acid residues while retaining these mutations, aprotein of which modified amino acid sequence having at least 78%homology with the amino acid sequence in (b), has a higher catalyticspecifity (namely selectivity) than the proline hydroxylases in priorart, or has remarkably improved catalytic activity when compared withthe wild-type hydroxylases (namely the proline hydroxylase having theamino acid sequence of SEQ ID NO: 2) discovered by the application.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings of the specification, which constitute a partof the application, are used for providing further understanding to thepresent application. The exemplary embodiments of the presentapplication and illustration thereof are used for explaining the presentapplication, instead of constituting improper limitation to the presentapplication. In the accompanying drawings:

FIG. 1 shows a chemical reaction of a use of a proline hydroxylaseaccording to the present application in catalytic-synthesizing(2S,5S)-5-hydroxypiperidine-2-carboxylic acid (or named ascis-5-hydroxypiperidine acid); and

FIG. 2 shows an equation of a chemical reaction of a use of a prolinehydroxylase according to the present application incatalytic-synthesizing cis-4-hydroxy-L-proline.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It needs to be noted that the embodiments in the invention and thecharacteristics in the embodiments may be combined with each other ifthere is no conflict. The present invention will be expoundedhereinafter with reference to the accompanying drawings and inconjunction with the embodiments.

Term Explanation

Selectivity of enzyme: or named as specifity of enzyme, it is theselectivity of the enzyme to a substrate in a catalytic biochemicalreaction. In the application, the selectivity of a proline hydroxylaserefers to the extent to which the proline hydroxylase catalyzeshydroxylation of an L-proline derivative to obtain a L-hydroxyprolinederivative of a particular configuration. In the reaction related to theapplication, the extent of catalytic selectivity of the hydroxylase maybe characterized with reacted diastereomeric excess.

Conversion rate: it is a percentage or a fraction of conversion of acertain reactant. The conversion rate is an index for representing areaction extent of the reactant. The conversion rate of hydroxylation ofL-piperidine acid in the application is a percentage of a product(2S,5S)-5-hydroxypiperidine-2-carboxylic acid generated in a reaction ofcatalyzing L-piperidine acid through hydroxylase or proline hydroxylaseaccounting for the L-piperidine acid in a system.

Catalytic activity: refers to the amount of reactant conversion per unitvolume (or mass) of catalyst per unit time. In the present application,the catalytic activity of the proline hydroxylase is positivelycorrelated with the conversion of the reaction.

Evolution: creating molecular diversity by means of mutations orrecombination, and screening the diversity to obtain a gene or DNA withnew functions. In the present application, the hydroxylase or theproline hydroxylase is modified through the means of mutations orrecombination, to obtain the hydroxylase or the proline hydroxylase withimproved performance.

Diastereoisomer: it is a stereoisomer of which molecules have two ormore chiral centers, and the molecules are in a non-mirroredrelationship.

Diastereomeric excess (de % for short): it is used for representingexcess of one diastereomer to other enantiomers in the two chiralcenters. Namely de %=(R,R+S,S)−(S,R+R,S)/(R,R+S,S+S,R+R,S).

Wild type: it is obtained from the nature, and is not artificiallymutagenized or modified. In the application, the wild type prolinehydroxylase is a natural proline hydroxylase screened from Genebank andis encoded by a gene sequence not artificially modified.

Homologous sequence: refers to a DNA sequence that is identical orsimilar between different individuals of the same species or thedifferent species.

In the application, related 1 wt refers to an 1 g proline hydroxylasevariant recombined wet cell needed for converting an 1 g main rawmaterial.

DISCUSSION

As mentioned in the background, a defect of the proline hydroxylase inthe prior art is that the catalytic selectivity is not high, anddifficulty is increased for separation of a follow-up target product. Inorder to solve the problem in the prior art that the selectivity of theproline hydroxylase is not high, inventors acquire a proline hydroxylasewith high catalytic specifity through genetic engineering. At the sametime, the inventors perform various mutation modifications on thewild-type proline hydroxylase in prior art, in order to obtain a prolinehydroxylase with high selectivity and improved catalytic activity.

The inventors screens countless homologous sequences of the amino acidsequence of the proline hydroxylase in prior art from Genebank,according to the order of homology from high to low, gene mutations indifferent positions were performed on various screened homologoussequences, and the hydroxylase activities of various mutants werescreened. It was discovered that the activity of the proline hydroxylaseof the mutant obtained by performing the mutation on the sequence withhigher homologous with the proline hydroxylase in prior art sequenceshas no apparent difference with the proline hydroxylase in prior art.Finally, only one sequence derived from Kordia jejudonensis which hasthe lowest homology (only about 30%) with the amino acid sequence of theproline hydroxylase in prior art was left, and the sequence has nospecific gene function annotation in the Genebank, and is annotated as ahypothetical protein. Recombination expression was performed on thesequence by means of genetic engineering, it was unexpectedly discoveredthat a protein encoded by the sequence has the activity of the prolinehydroxylase, and has the selectivity higher than that of the prolinehydroxylase in the prior art. The protein is named as the wild-typeproline hydroxylase by the inventors. The application further performs amutation test on the amino acid sequence of the wild-type prolinehydroxylase, it is more surprised that the mutant obtained by themutation of the sequence derived from Kordia jejudonensis not only hasthe activity of the proline hydroxylase, but also the catalytic activitythereof is remarkably improved compared with the wild-type prolinehydroxylase.

Further, the inventors perform various mutation screening based on thesequence, and finally it was discovered that multiple mutation sites arerelated to the activity of the proline hydroxylase. Under theprecondition of retaining these mutation sites, the activity of theproline hydroxylase is not apparently affected by arbitrarily changingthe mutations of other sites.

On the above basis, the inventors performed a deeper research, and someimportant amino acid residue sites were discovered, after the mutationof the sites, soluble expression of the proline hydroxylase thereof isdecreased significantly or the catalytic activity of the prolinehydroxylase is lost. After S3, L94, H105, D107, S131, E132, Y137, M139,W145, H153, N157, V167, D169 amino acid residue sites are mutated to theother majority of amino acids, the soluble expression of the prolinehydroxylase is decreased significantly, some are zero even. Themutations of these amino acids affect folding of the prolinehydroxylase, so the soluble expression of the proline hydroxylase in anE. coli host cell is significantly decreased, and even the solubleexpression is decreased to zero. The mutated amino acid of the prolinehydroxylase may be selected from: Y35A, Y35F, Y355, W40Y, L94A, L94G,L945, F95A, F95W, H105R, H105Q, H105E, H105G, R117A, R117P, R117K,P121V, V135A, S131T, H153Y, H153A, H153R, H153K. At the same time, it isfurther discovered that after the Y32, R93, R117, Y108 amino acidresidue sites are mutated to the other majority of the amino acids, thecatalytic activity of the proline hydroxylase is lost. These mutatedamino acids which are capable of significantly decreasing, even losingthe catalytic activity of proline hydroxylase may be selected from:Y32N, Y32V, Y32V, Y32Q, Y32E, Y32S, Y32R, Y32D, Y32R, Y321, Y32P, Y35H,Y35F, Y35A, W40Y, R93K, R93H, R93E, R93A, L94A, F95W, F95A, H105E,H105Q, H105R, H105K, H105A, K106H, D107A, Y108W, Y108A, Y108L, Y108S,R117D, R117P, R117N, R117N, R117H, R117K, R117A, P121V, H153W, H153F,H153K, H153R, H153A.

Computer simulation was performed on a three-dimensional structure ofthe proline hydroxylase, and a simulated three-dimensional structurediagram was obtained. After the structure diagram was analyzed, it wasspeculated that most of these sites should be the important amino acidresidues for participating in combination of the proline hydroxylasewith a substrate or combination with a cofactor, for example, H105,D107, and H153 may participate in the combination of the cofactor withthe proline hydroxylase. And for example, Y32, R93, and R117 mayparticipate in the combination of the substrate with the prolinehydroxylase, herein R93 and R117 amino acid residues may form a saltbridge with the substrate. In addition, the amino acid residues at230-276 are outside an activity relevant area of the enzyme, somodifying or removing some of the amino acids in the area does not havea significant effect on the activity of the proline hydroxylase.

The mutants of the application are the mutants with improved catalyticactivity or selectivity which are obtained by modifying the rest aminoacids in the case of keeping the above amino acid sites havingsignificant effects on the activity of the proline hydroxylaseunchanged.

On the basis of the above research results, the inventors provide thetechnical solution of the application. In a typical embodiment, aproline hydroxylase is provided, wherein the proline hydroxylasecomprises: (a) a protein having an amino acid sequence as shown in SEQID NO: 2; (b) a protein having an amino acid sequence of SEQ HD NO: 2with a mutation of one or more amino acids and having a prolinehydroxylase activity; or (c) a protein retaining the mutation of one ormore amino acids as in (b), and having the proline hydroxylase activityand having at least 78% homology with the amino acid sequence of theprotein in (b).

The above proline hydroxylase, through selecting SEQ ID NO:2 as a basesequence, a mutant containing single or multiple amino acid residuesmodified through genetic engineering, or by altering other amino acidresidues while retaining these mutations, a protein of which modifiedamino acid sequence having at least 78% homology with the amino acidsequence in (b), has a higher catalytic specifity (namely selectivity)than the proline hydroxylase in prior art, or has remarkably improvedcatalytic activity when compared with the wild-type hydroxylase (namelythe proline hydroxylase having the amino acid sequence of SEQ ID NO: 2)discovered by the application.

The catalytic activity of the above proline hydroxylase is at leastimproved by 1 time, 2 times, 3 times, 4 times, 5 times or more comparedwith the original wild type hydroxylase encoded by the SEQ ID NO:2. Inaddition, the selectivity of the above proline hydroxylase is remarkablyimproved compared with the prior art, the diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid catalytically generated bythe proline hydroxylase is greater than 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more.

In some embodiments, the above proline hydroxylase is the protein havingthe amino acid sequence of SEQ ID NO:2 with the mutation of one or moreof the following amino acid sites and having the proline hydroxylaseactivity: H14, S16, T25, F26, E27, D30, S33, E34, Y35, S37, I39, W40,K41, D54, H55, S57, I58, K86, T91, F95, C97, I98, K106, F111, K112,K162, L166, I118, H119, I120, K123, T130, D134, V135, S154, N165, D173,K209, I223 and S225. Wherein, the amino acid sites related to theproline hydroxylase activity are as follows: H14, S16, F26, E27, D30,S33, E34, S37, I39, W40, K41, T91, F95, C97, I98, K106, F111, K112,I118, H119, I120, K123, T130, D134, V135, N165, K209 and I223. Inaddition, Y35 and S57 amino acid sites relate to the selectivity of thehydroxylase. The mutation of the above one or more amino acid sites hasremarkable effects on the activity or selectivity of the wild typeproline hydroxylase, and is capable of remarkably improving the prolinehydroxylase catalytic activity and/or selectivity of the mutant.

The three-dimensional structure simulated diagram of the above prolinehydroxylase was further analyzed, it was speculated that the activesites of the proline hydroxylase in the application are located in aBeta folding related region, and the region is fixed by an A helicalstructure positioned in the N-terminal and C-terminal. The substratecombination site is positioned in a center of the Beta folding area, andadjacent to a cofactor combination area. These sites are analyzed, andit was discovered that the mutated amino acid residues of the prolinehydroxylase referred in the application are mainly positioned in thesubstrate combination sites or the region related to the cofactorcombination in the three-dimensional structure simulated diagram of theproline hydroxylase. For example, E27, D30, S33, E34, Y35, S37, I39,W40, K41, H55, S57, I58, F95, C97, I98, F111, K112, I118, H119, and I120amino acids may be positioned near the substrate combination sites, andspecificity of the substrate combination may be improved through themodification of these amino acids, so the activity or catalyticselectivity of the enzyme is improved. For example, K106, L166, K123,D134, S154, N165 amino acids may be positioned near the cofactorcombination sites, the modification of these amino acids may improve thecombination of the cofactor, and coordinate the utilization andtransmission of oxygen, so the activity of the enzyme is improved.

On the basis of the mutation of the above sites, through mutating thesesites to different amino acids and detecting the change of the activityof the proline hydroxylase thereof, it was discovered that after theseamino acid sites are mutated to any one or more of the followingcombinations, the activity and/or selectivity of the hydroxylase isfurther improved. The mutation comprises any one or more of thefollowings: H14R, 516N, T25G, T25R, F26L, E27K, D30S, S33N, E34N, E34G,E34L, E34S, E34D, Y35W, Y35K, S37W, S37F, S37E, S37N, S37T, S37C, I39K,139R, W40F, K41E, D54G, H55Q, S57L, I58T, I58Y, 158A, I58R, I58V, I58S,I58C, K86P, T91A, F95Y, C97Y, I98V, K106V, K106T, K106Q, F111S, K112E,K112R, S154A, K162E, L166M, I118F, I118V, I118R, H119R, H119F, I120V,K123D, K123N, K123Q, K123S, K123I, K123T, T130N, D134G, V135K, N165H,D173G, K209R, I223V and S225A. More preferably, the mutation comprisesany one or more of the followings: H14R, E27K, E34N, E34G, E34L, E34D,Y35W, Y35K, S37W, S37F, S37E, S37N, S37T, S37C, I39K, 139R, I58T, I58Y,I58A, I58R, I58V, I58S, I58C, K123D, K123N, K123Q, K123S, K123I andK123T. Herein, the amino acid mutations related to the selectivity ofthe proline hydroxylase are as follows: Y35W, S57L and S57V.

The mutation of some amino acids may improve the soluble expressionquantity of the proline hydroxylase in a bacterial cell, especially thesoluble expression in an E. Coli host cell, these mutated amino acidsmay be selected from: E27K, D30S, Y35W, Y35K, S37W, S37F, S37E, S37N,S37T, S37C, I39K, 139R, W40F, I58T, I58Y, I58A, I58R, I58V, I58S, I58C,I98V, K106V, K106T, K106Q, H119R, H119F, K123D, K123N, K123Q, K123S,K123I, K123T, N165H, I223V. Generally, when an exogenous gene isexpressed in a prokaryotic expression system, only the soluble proteincorrectly folded is active, a formed inclusion body is inactive. Thesoluble protein expression quantity is increased, and the total enzymeactivity is increased accordingly.

In a more preferable embodiment, the above mutation comprises any one ofthe following combinations: E27K+Y35W/K, E27K+I39K/R, E27K+K123D/I/Q/S,E27K+N165H, I39K/R+Y35W/K, I39K/R+K123D/I/Q/S, I39K/R+N165H, K123D+W40F,K123D+Y35W/K, E27K+I39K/R+K123D/I/Q/S, K123D/I/Q/S+N165H,S37C/E/F/N/W/T+I223V, E27K+Y35W/K+I39K/R, E27K+S37C/E/F/N/W/T+I39K/R,E27K+E34N/G/L/D/S+I39K/R, E27K+I39K/R+D30S, E27K+I39K/R+I118 F/V/R,E27K+I39K/R+I98V, S37C/E/F/N/W/T+I223V+N165H,Y35W/K+S37C/E/F/N/W/T+W40F, S37C/E/F/N/W/T+I223V+K123D/I/Q/S,E27K+I39K+Y35W/K+S37C/E/F/N/W/T, E27K+I39K/R+S37C/E/F/N/W/T+K123D/I/Q/S,E27K+I39K/R+K106Q+K112E, E27K+I39K/R+Y35W/K+S37C/E/F/N/W/T+K123D/I/Q/S,E27K+I39K/R+S37C/E/F/N/W/T+I58A/C/R/S/T/V/Y,E27K+S37C/E/F/N/W/T+I223V+K123D/I/Q/S,S37C/E/F/N/W/T+I39K/R+I223V+K123D/I/Q/S,E27K+S37C/E/F/N/W/T+I39K/R+K123D/I/Q/S+I98V,E27K+S37C/E/F/N/W/T+I39K/R+K123D/I/Q/S+I223V,F26L+E27K+I39K/R+K123D/I/Q/S, I223V+S37C/E/F/N/W/T+E27K+I39K/R,I223V+S37C/E/F/N/W/T+E27K+N165H,E27K+S37C/E/F/N/W/T+I39K/R+I98V+K123D/I/Q/S+I223V, K106Q+K112E+I223V,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+K123D/I/Q/S,E27K+I39K/R+K123D/I/Q/S+N165H, H14R+E34G+K106Q+K112E+I223V,T25G/R+E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+K86P,K123D/I/Q/S+Y35W/K+I120V, E27K+D30S+I39K/R+I58A/C/R/S/T/V/Y+K112E,S37C/E/F/N/W/T+I39K/R+N 165H,E27K+E34N/G/L/D/S+I39K/R+I58A/C/R/S/T/V/Y+I223V,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+I118F/V/R,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/I/Q/S,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/I/Q/S+I118F/V/R,E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/I/Q/S+N165H,E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/+D173G+K123D/I/Q/S,H14R+E27K+D30S+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,T25G/R+E27K+S37C/E/F/N/W/T+I39K/R+I58A/C/R/S/T/V/Y+D173G+K123D/1/Q/S+I118F/V/R+N165H, H14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+S37C/E/F/N/W/T+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+Y35W/K+I39K/R+I98V+K106V/T/Q+K112E/R+I223V,H14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+K123D/I/Q/S+I223V,H14R+E27K+E34N/G/L/D+I39K/R+I58A/C/R/S/T/V/Y+I98V+K106V/T/Q+K112E/R+I223VandH14R+E27K+E34N/G/L/D/S+I39K/R+I98V+K106V/T/Q+K112E/R+I118F/V/R+I223V.‘/’ in the above mutation combinations stands for ‘or’. In the abovemutation combinations, the combinations referring to the mutation ofY35W are related to the selectivity of the proline hydroxylase, and theother combinations are related to the catalytic activity of the prolinehydroxylase.

In the above preferable embodiment, catalytic activity information ofthe proline hydroxylase is screened on the basis of the catalyticactivity of the enzyme to L-piperidine acid. Catalytic activity resultsof the proline hydroxylase in these more preferable embodiments are asshown in Table 1 and Table 2, a sequence number of the DNA sequence isan odd, and a sequence number of the amino acid sequence is an even. Themutated amino acid of the application is obtained by modification basedon the amino acid sequence of SEQ ID NO: 2, and the SEQ ID NO: 2 is thesequence of hypothetical protein derived from Kordia jejudonensis. Theactivity of the wild type proline hydroxylase in the application isrepresented by ‘1’, the activity of the mutant proline hydroxylase isrepresented by ‘+’: ‘+’ represents that the activity is 1-2 times ofwild type SEQ ID NO:2, ‘++’ represents that the activity is 2-3 times ofwild type SEQ ID NO:2, ‘+++’ represents that the activity is 3-4 timesof wild type SEQ ID NO:2, and ‘++++’ represents that the activity is 4-5times of wild type SEQ ID NO:2.

TABLE 1 Comparison of proline hydroxylase activity after mutation of asingle site. SEQ ID NO: Mutated amino No. (DNA/AA) acid Activity 1 1/2

1 2 3/4

1 3 5/6 H14R + 4 7/8 S16N + 5  9/10 T25G + 6 11/12 T25R + 7 13/14 F26L +8 15/16 E27K + 9 17/18 D30S + 10 19/20 S33N + 11 21/22 E34N + 12 23/24E34G + 13 25/26 E34L + 14 27/28 E34D + 15 29/30 E34S + 16 31/32 Y35W ++17 33/34 Y35K ++ 18 35/36 S37W + 19 37/38 S37F + 20 39/40 S37E + 2141/42 S37N + 22 43/44 S37T + 23 45/46 S37C + 24 47/48 I39K ++ 25 49/50I39R ++ 26 51/52 W40F + 27 53/54 K41E + 28 55/56 D54G + 29 57/58 H55Q +30 59/60 S57L + 31 61/62 I58T + 32 63/64 I58Y + 33 65/66 I58A + 34 67/68I58R + 35 69/70 I58V + 36 71/72 I58S + 37 73/74 I58C + 38 75/76 K86P +39 77/78 T91A + 40 79/80 F95Y + 41 81/82 C97Y + 42 83/84 I98V + 43 85/86K106V + 44 87/88 K106T + 45 89/90 K106Q + 46 91/92 F111S + 47 93/94K112E + 48 95/96 I118F + 50 97/98 H119R ++ 51  99/100 H119F + 52 101/102I120V + 53 103/104 K123D ++ 54 105/106 K123N + 55 107/108 K123Q + 56109/110 K123S + 57 111/112 K123I + 58 113/114 K123T + 59 115/116 T130N +60 117/118 D134G + 61 119/120 V135K + 62 121/122 N165H + 63 123/124K209R + 64 125/126 I223V +

The Table 1 provides the effects on the catalytic activity of themutated proline hydroxylase by performing the modification of the aminoacids at different sites on the basis of the sequence of SEQ ID NO:2.Herein, the mutated proline hydroxylase was expressed from an E. coliBL21 cell, the catalytic activity was based on conversion efficiency ofthe enzyme to L-piperidine acid. The above catalytic reaction wasperformed in a 10 ml reaction system, and the reaction system comprises:30 g/L L-piperidine acid, 5-10 wt recombinase (1 wt is an 1 g prolinehydroxylase variant recombined wet cell needed for converting an 1 gmain raw material), 37.3 g/L α-ketoglutarate, 6.1 g/L L-ascorbic acid, 5mM ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours.

TABLE 2 Comparison of proline hydroxylase activity after multi-sitemutation. SEQ ID NO: No. (DNA/AA) Mutated amino acid Activity 1 127/128E27K + K123D ++ 2 129/130 E27K + I39K +++ 3 131/132 I39K + K123Q ++ 4133/134 I39K + K123D ++ 5 135/136 S37C + I223V + K123D ++ 6 137/138K123D + N165H ++ 7 139/140 I39R + K123D +++ 8 141/142 E27K + I39K +K123D +++ 9 143/144 E27K + I39R +++ 10 145/146 I39R + N165H + 11 147/148I39K + N165H + 12 149/150 E27K + I39R + K123D +++ 13 151/152 S37C +I223V + N165H + 14 153/154 K123D + W40F ++ 15 155/156 E27K + N165H ++ 16157/158 E27K + S37C + I223V + K123D +++ 17 159/160 S37C + I39K + I223V +K123D ++++ 18 161/162 E27K + S37C + I39K + K123D + I98V ++++ 19 163/164E27K + S37C + I39K + K123D + I223V ++ 20 165/166 F26L + E27K + I39K +K123D ++ 21 167/168 I223V + S37C + E27K + I39K +++ 22 169/170 I223V +S37C + E27K + N165H +++ 23 171/172 K123D + Y35W ++ 24 173/174 K123D +Y35W + I120V ++ 25 175/176 E27K + Y35W ++ 26 177/178 I39R + Y35W ++++ 27179/180 S37C + I223V ++ 28 181/182 E27K + I39K + Y35W ++++ 29 183/184E27K + S37C + I39K + I98V + K123D + I223V ++ 30 185/186 E27K + I39K +K123D + N165H + 31 187/188 E27K + I39R + Y35W +++ 32 189/190 S37C +I39K + N165H ++ 33 191/192 E27K + I39K + D30S +++ 34 193/194 E27K +I39K + E34N +++ 35 195/196 E27K + I39K + E34G +++ 36 197/198 E27K +I39K + S37W +++ 37 199/200 E27K + I39K + S37E +++ 38 201/202 E27K +I39K + Y35K +++ 39 203/204 E27K + I39K + S37N +++ 40 205/206 E27K +I39K + K123Q +++ 41 207/208 E27K + I39K + K123S +++ 42 209/210 E27K +I39K + K106Q + K112E +++ 43 211/212 I98V + E27K + I39K +++ 44 213/214E27K + I39K + I118F +++ 45 215/216 E27K + I39K + S37T +++ 46 217/218E27K + I39K + K123I +++ 47 219/220 K106Q + K112E + I223V +++ 48 221/222H14R + E34G + K106Q + K112E + I223V +++ 49 223/224 E27K + I39K + E34L+++ 50 225/226 E27K + I39K + S37F + I58T +++ 51 227/228 E27K + I39K +S37F + I58Y +++ 52 229/230 E27K + I39K + S37F + I58A +++ 53 231/232E27K + I39K + S37F + I58R +++ 54 233/234 E27K + I39K + S37F + I58V +++55 235/236 E27K + I39K + S37F + I58S +++ 56 237/238 E27K + I39K + S37N +I58C +++ 57 239/240 E27K + D30S + I39K + I58R + K112E ++++ 58 241/242E27K + E34N + I39K + I58Y + I223V ++++ 59 243/244 E27K + S37N + I39K +I58Y + D173G +++ 60 245/246 E27K + S37F + I39K + I58Y + D173G +++ 61247/248 E27K + I39K + S37N + I58A +++ 62 249/250 E27K + I39K + S37N +I58R + K123Q +++ 63 251/252 E27K + S37F + I39K + I58Y + D173G + I118R+++ 64 253/254 E27K + E34L + S37N + I39K + I58R +++ 65 255/256 E27K +E34L + S37N + I39K + I58Y + D173G +++ 66 257/258 E27K + E34L + S37N +I39K + I58Y + D173G + +++ K123Q 67 259/260 H14R + E27K + D30S + E34G +I39K + I98V + +++ K106Q + K112E + I223V 68 261/262 H14R + E27K + E34N +I39K + I98V + K106Q + +++ K112E + I223V 69 263/264 H14R + E27K + E34G +I39K + I98V + K106Q + +++ K112E + I223V 70 265/266 H14R + E27K + E34G +S37W + I39K + I98V + +++ K106Q + K112E + I223V 71 267/268 H14R + E27K +E34G + S37F + I39K + I98V + +++ K106Q + K112E + I223V 72 269/270 H14R +E27K + E34G + S37E + I39K + I98V + +++ K106Q + K112E + I223V 73 271/272H14R + E27K + E34G + Y35K + I39K + I98V + ++++ K106Q + K112E + I223V 74273/274 H14R + E27K + E34G + S37N + I39K + I98V + +++ K106Q + K112E +I223V 75 275/276 H14R + E27K + E34G + I39K + I98V + K106Q + ++++ K112E +K123Q + I223V 76 277/278 H14R + E27K + E34G + I39K + I98V + K106Q + +++K112E + K123S + I223V 77 279/280 H14R + E27K + E34G + I39K + I58A +I98V + +++ K106Q + K112E + I223V 78 281/282 H14R + E27K + E34G + I39K +I98V + K106Q + +++ K112E + I118F + I223V 79 283/284 H14R + E27K + E34G +S37T + I39K + I98V + +++ K106Q + K112E + I223V 80 285/286 H14R + E27K +E34G + I39K + I58V + I98V + +++ K106Q + K112E + I223V 81 287/288 H14R +E27K + E34G + I39K + I98V + K106Q + +++ K112E + K123I + I223V

The Table 2 provides the effects on the catalytic activity of theproline hydroxylase through the modification of amino acids at multiplesites. The proline hydroxylase was expressed from the E. Coli BL21 cell,the catalytic activity was based on the conversion efficiency of theenzyme to the L-piperidine acid. The catalytic process was performed ina 20 ml reaction system, herein the reaction system comprises 50 g/LL-piperidine acid, 2-5 wt recombinase, 62.2 g/L α-ketoglutarate, 10.2g/L L-ascorbic acid, 5 mM ammonium ferrous sulfate, the reaction pH was6.5, the reaction temperature was 10 DEG C., and the reaction time was40 hours.

In some embodiments, one or more amino acid residues related to theproline hydroxylase activity is selected as a core and kept unchanged,and new mutation is introduced in other amino acid residue positions,the proline hydroxylase with the improved property may be generated. So,any one proline hydroxylase in the above preferable embodiments may beused as a female parent amino acid sequence for synthesizing otherproline hydroxylase mutants by genetic engineering. For example newmutants of which the amino acid residues obtained by several rounds ofevolution are different from the amino acid sequences in the Table 1 andTable 2.

Any one of the above disclosed proline hydroxylases, or any new mutanthaving the proline hydroxylase activity obtained by performing themutation of one or more amino acid residues in the other amino acidresidue positions on the basis of the above disclosed prolinehydroxylases or the variants thereof are within the scope of protectionof the application. It may be illustrated, but not limited to this, theproline hydroxylase mutant containing the mutation of the E27 amino acidresidue may further be performed the mutation of one or more other aminoacids, for example: H14, S16, T25, F26, D30, S33, E34, Y35, S37, I39,W40, K41, D54, H55, S57, I58, K86, T91, F95, C97, I98, K106, F111, K112,K162, L166, I118, H119, I120, K123, T130, D134, V135, S154, N165, D173,K209, I223, S225. Another example is that the proline hydroxylase mutantcontaining the mutation of the 139 amino acid residue may further beperformed the mutation of one or more other amino acids, for example:H14, S16, T25, F26, E27, D30, S33, E34, Y35, S37, W40, K41, D54, H55,S57, I58, K86, T91, F95, C97, I98, K106, F111, K112, K162, L166, I118,H119, I120, K123, T130, D134, V135, S154, N165, D173, K209, I223, S225.Another example is that the proline hydroxylase mutant containing themutation of the 158 amino acid residue may further be performed themutation of one or more other amino acids, for example: H14, S16, T25,F26, E27, D30, S33, E34, Y35, S37, I39, W40, K41, D54, H55, S57, I58,K86, T91, F95, C97, I98, K106, F111, K112, K162, L166, I118, H119, I120,K123, T130, D134, V135, S154, N165, D173, K209, I223, S225.

The above hydroxylases have the proline hydroxylase activity, and arecapable of catalyzing (2S)-piperidine-2-carboxylic acid to be convertedto (2S,5S)-5-hydroxypiperidine-2-carboxylic acid, and improving thecatalytic activity of the enzyme through genetic engineering, thehydroxylase activity of the mutants in some embodiments is improved by 1time, 2 times, 3 times, 4 times, 5 times or more compared with theactivity of the hydroxylase encoded by SEQ ID NO: 2 itself.

In another typical implementation mode of the application, a DNAmolecule is provided, wherein the DNA molecule encodes any one of theabove hydroxylases. The encoded hydroxylases have the advantages of highspecifity and remarkably improved catalytic activity.

In another typical implementation mode of the application, a recombinantvector is further provided, wherein the recombinant vector is connectedwith the DNA molecule. The DNA molecule may encode any one of the aboveproline hydroxylases with high selectivity, and/or remarkably improvedcatalytic activity. The specific sequence is selected from the sequencein Table 1 and Table 2 of which the number is an odd, or a nucleotidesequence which is generated by substitution, addition or deletionmutation within the amino acid sequences in the other sites in theprecondition of keeping the changed amino acid sites of these sequences.

In the above recombinant vector, any recombinant vector which may beused for expressing the DNA molecule of the above hydroxylase aresuitable for the application. In the preferable embodiment of theapplication, the recombinant vector is selected from one of thefollowings: pET-22b(+), pET-21b(+), pET-3a(+), pET-3d(+), pET-11a(+),pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+),pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+),pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+),pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+),pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+),pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B,pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222,pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18 and PUG 19.

In another typical implementation mode of the application, a host cellis provided, wherein the host cell comprises any one of the recombinantvectors. The specific host cell may be a prokaryotic cell or aeukaryocyte, preferably the eukaryocyte is a yeast cell. Morepreferably, the host cell is a competent cell, further preferably thecompetent cell is an E. coli BL21 cell or an E. coli W3110 cell.

In another typical implementation mode of the application, a method forproducing an L-hydroxyproline derivative is further provided, whereinthe method comprises the following steps: using an L-proline derivativeas a substrate, and applying the proline hydroxylases as claimed in anyone of claims 1 to 4 to catalyze hydroxylation of the substrate, toobtain the L-hydroxyproline derivative as shown in a general formula(I):

wherein R₁ is selected from C₁-C₅ alkylene or C₂-C₅ alkenylene; R₂ isselected form C₀-C₄ alkylene or C₂-C₄ alkenylene; R₃ is selected fromhydroxyl, amino, C₁-C₆ alkoxy, aryloxy, C₁-C₆ alkyl sulfenyl or C₁-C₆aryl sulfenyl; and R₄ is selected from hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl or C₂-C₆ alkynyl.

The above proline hydroxylase of the application is used for catalyzingthe L-proline derivative to be a hydroxide thereof, not only catalyticefficiency may be improved, and the conversion rate of the L-prolinederivative is improved, but also specifity of the catalysis is improved,so a purity of an obtained target product of the L-hydroxyprolinederivative is improved, and the follow-up separating process is reduced.

The chemical reaction equation for catalytic-synthesizing theL-hydroxyproline derivative with the proline hydroxylase is shown inFIG. 2 . Herein,

is α-ketoglutarate, in the catalytic reaction of the prolinehydroxylase, the α-ketoglutarate and O₂ are used as a common substrate.Under the combined action of iron ions as a cofactor, the hydroxylationof the substrate is catalyzed, to obtain the L-hydroxyprolinederivative;

is vitamin C, named as ascorbic acid too, in the catalytic reaction ofthe proline hydroxylase, which mainly plays the role of circulating theiron ions.

As a typical dioxygenase, just like the proline hydroxylase in priorart, in the catalytic reaction of the proline hydroxylase,α-ketoglutarate and the O₂ are needed, and the iron ions are needed asthe cofactor. So the method comprises: the α-ketoglutarate and the O₂are used as the common substrate, the iron ions are used as thecofactor, the hydroxylation of the substrate is catalyzed by the prolinehydroxylase, so the L-hydroxyproline derivative is obtained.

The specific reaction conditions may be appropriately adjusted on thebasis of the proline hydroxylase reaction system in prior art. Forexample, a concentration of a reducing agent (for example, the ascorbicacid), a concentration of a detergent, pH value, temperature, buffering,a solvent system, substrate loading, polypeptide loading, a pressure andreaction time and the like may be appropriately adjusted. In someembodiments, the specific reaction conditions are as follows: 15˜120 g/Lof the substrate, 1.5˜48 g/L of the hydroxylase, 1˜2.5 eq (eq:represents a proportion value of a mass of a used material and a mass ofa main raw material) of the α-ketoglutarate, 0.1˜0.3 eq of L-ascorbicacid, 1˜10 mM of the ammonium ferrous sulfate, 6˜8 of the reaction pH,5˜30 DEG C. of the reaction temperature, and 6˜96 hours of the reactiontime. In some embodiments, the appropriate reaction condition comprisesthat 2˜5 L/h of oxygen and 0.5˜2% of a defoaming agent are fed into thereaction solution.

In a preferably embodiment of the application, the proline hydroxylasemay catalyze the hydroxylation of the substrate at 5˜45 DEG C. of thereaction temperature, and the L-hydroxyproline derivative as shown inthe general formula I is obtained. More preferably, the catalyticreaction is performed at 5˜15 DEG C. of the reaction temperature. Theimproved proline hydroxylase of the application may not only catalyzethe substrate to the hydroxide thereof in the lower temperature, butalso improve the catalytic specifity (namely the selectivity), and thepurity of the product is improved.

The enzyme may perform hydroxylation on multiple types of the L-prolinederivatives. In a preferable embodiment of the application, thehydroxylation is performed on the L-proline or L-piperidine acid,cis-4-hydroxy-L-proline or (2S,5S)-5-hydroxypiperidine-2-carboxylic acidis obtained. The specifity of the hydroxylation to the above twosubstrates is the highest, and 100% of the substrate may be converted tothe cis-4-hydroxy-L-proline or the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid.

The beneficial effects of the application are further described below incombination with the specific embodiments. Experimental methods beloware conventional methods if not specifically indicated, and usedexperimental materials may easily be acquired from a commercialcorporation if not specifically indicated.

Embodiment 1: Recombination Expression of Proline Hydroxylase

Codon optimization (codon improvement was performed according to codonbias and degeneracy for E. coli, which was designed and completed byGENEWIZ SuZhou Co., Ltd) was performed on a DNA coding sequence SEQ IDNO: 1 annotated as a hypothetical protein and derived from Kordiajejudonensis, and the optimized DNA sequence SEQ ID NO: 3 was obtained,and the encoded proline hydroxylase polypeptide sequence is SEQ ID NO:4. The coding sequence of SEQ ID NO: 3 was connected to a pET22b(+)expression vector (purchased from Novagen, and the product number is69744), and transformed into E. coli BL21 (DE3), coated in an LB culturedish containing ampicillin having a final concentration of 50 μg/ml, andcultured overnight at 37 DEG C. A single colony on the culture dish wasselected and inoculated in 500 ml of an LB liquid culture mediumcontaining ampicillin having a final concentration of 50 μg/ml, thencultured by shaking at 37 DEG C. until OD₆₀₀=0.6, IPTG was added untilthe final concentration being 1 mM, and induced to express at 25 DEG C.After induction for 16 hours, the thalli were collected bycentrifugation at 6000 g for 10 min. The thalli were disrupted by anultrasonic cell disruptor (JY92-2D, Ningbo Xin Zhisheng science andtechnology Co., Ltd), and the supernatant was obtained by centrifugationat 10000 g for 20 min at 4 DEG C. for detection of wild-type prolinehydroxylase activity as a control for screening the mutant activity.

Embodiment 2: Preparation of Proline Hydroxylase Mutants

A pET22b (+) expression vector containing the sequence of SEQ ID NO: 3was used as a template, and a primer with a mutation site was used foracquiring a complete linear fragment through a full-length plasmid PCR,and the PCR product was digested by DPn I to remove the female parenttemplate, and then transformed into E. coli BL21(DE3), coated in an LBculture dish containing ampicillin having a final concentration of 50μg/ml and incubated overnight at 37 DEG C., and a monoclone containingan amino acid sequence of the proline hydroxylase mutant was obtained,and the mutation site was determined through induction testing and genesequencing. Finally mutants with single mutation sites were obtained,and the mutants with the single mutation sites were used as a mutatedfemale parent, and the primers with a mutation in other sites were usedfor performing the full-length plasmid PCR again, and then mutationsites were detected again.

After activated, the mutant bacteria was inoculated into 500 ml of LBfluid culture medium containing ampicillin having a final concentrationof 50 μg/ml, and subjected to shake culture at 37 DEG C. untilOD₆₀₀=0.6, then IPTG was added to a final concentration of 1 mM, andinduction expression was carried out at 25 DEG C. After induction for 16hours, the cells were collected by centrifugation at 6000 g for 10 min.The thalli were disrupted by an ultrasonic cell disruptor (JY92-2D,Ningbo Xin Zhisheng science and technology Co., Ltd), and thesupernatant was obtained by centrifugation at 10000 g for 20 min at 4DEG C. for activity detection of proline hydroxylase variants.

Embodiment 3: Activity Screening of Proline Hydroxylase Variants

The activity screening of the proline hydroxylase variants in which oneamino acid residue differs from SEQ ID NO: 2 was screened using thefollowing 10 mL of the reaction solution, and the 10 mL of the reactionsolution comprises: 30 g/L of L-piperidine acid, 5-10 wt of recombinantcrude enzyme (1 wt is 1 g proline hydroxylase variant recombinant wetcell needed for converting an 1 g main raw material), 37.3 g/L ofα-ketoglutarate, 6.1 g/L of L-ascorbic acid, 5 mM of ammonium ferroussulfate, the reaction pH was 6.5, the reaction temperature was 10 DEGC., and the reaction time was 40 hours. At the end of the reaction, 200μL of the reaction system was taken, and then 200 μL of acetonitrile wasadded thereof, and 3000 μL of purified water was added after uniformlymixing, and the supernatant was collected by centrifugation at 10000 rpmfor 5 min for HPLC to determine the conversion rate. The activityscreening results were shown in Table 1 (Table 1 shows the activityscreening results obtained according to the comparison of all conversionrate data).

The activity screening of the proline hydroxylase variants in whichmultiple amino acid residues differ from SEQ ID NO: 2 was screened usingthe following 20 mL of reaction solution, and the 20 mL of the reactionsolution comprises: 50 g/L of L-piperidine acid, 2-5 wt of recombinantcrude enzyme, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid,5 mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, then 200μL of acetonitrile was added thereof, and 3000 μL of purified water wasadded after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate, and the activity screening results were shown in Table2 (Table 2 shows the activity screening results obtained according tothe comparison of all conversion rate data).

Embodiment 4: Clone and Expression of Proline Hydroxylase Mutants

In order to conveniently express and identify the hydroxylase mutants, acompatible restriction site was designed at 5′ and 3′ terminals of thegene thereof. Nde I and Xho I may be used for simultaneously performingdigestion on the target gene and pET-22b(+) (other expression plasmidswhich may express the protein in E. coli may be also used), the targetgene after the digestion and a larger fragment of the plasmid wereperformed a ligation reaction with a T4 DNA ligase, and a ligationproduct was converted into competent cells of E. coli DH5a strains, andthe converted competent cells were coated on an LB culture platecontaining ampicillin with a final concentration of 50 μg/ml, andcultured overnight at 37 DEG C.

A single colony grown on the above culture dish was selected, andinoculated in an LB fluid culture medium containing ampicillin having afinal concentration of 50 μg/ml, and subjected to shake culture at 37DEG C. overnight, bacteria liquid was collected, and after plasmidextraction, PCR identification and double digestion identification wereperformed, a correct clone vector was named as pET22b (+)-R-M andtransformed into E. coli BL21(DE3). The transformed E. coli BL21(DE3)was coated on the LB culture plate containing ampicillin having a finalconcentration of 50 μg/ml, and cultured at 37 DEG C. overnight. Thesingle colony grown on the above culture plate was selected, andinoculated in 5 ml of the LB fluid culture medium containing ampicillinhaving a final concentration of 50 μg/ml, and the colony PCR was usedfor identification, and the E. coli cells containing the correctexpression vector were performed the follow-up induction expression. Theabove bacteria solution is transferred and inoculated in 500 ml of theLB fluid culture medium containing ampicillin having a finalconcentration of 50 μg/ml, then cultured by shaking at 37 DEG C. untilOD₆₀₀=0.5-0.6, IPTG was added until the final concentration being0.2-1.0 mM, after the induced expression was performed at 18-25 DEG C.for 10-16 hours, the bacteria liquid was taken out, and the thalli werecollected and centrifuged at 6000 g for 10 min, and frozen-stored forfuture use in −20 DEG C. The thalli were broken by an ultrasonic celldisruptor (JY92-2D, Ningbo Xin Zhisheng science and technology Co.,Ltd), and the supernatant and precipitate were obtained at 4 DEG C. bycentrifugation at 10000 g for 20 min, and the supernatant were detectedby SDS-PAGE with a vertical electrophoresis apparatus. The molecularweight of the expressed hydroxylase mutant displayed on SDS-PAGE wasabout 30 KD.

Embodiment 5: Performance Comparison of Hydroxylases in Table 1 andTable 2 and Wild Type Proline Hydroxylase

The following experiments were performed according to the chemicalreaction process as shown in FIG. 1 for hydroxylase-catalyzed synthesisof (2S,5S)-5-hydroxypiperidine-2-carboxylic acid (or named ascis-5-hydroxypiperidine acid):

20 ml of the following reaction solution was used in a process that thewild type proline hydroxylase encoded by SEQ ID NO:2 catalyzedL-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 10 wt of hydroxylase,62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5 mM ofammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. At 40 hours, a conversion rate was 98.47%, anddiastereomeric excess of the (2S,5S)-5-hydroxypiperidine-2-carboxylicacid was 98.36%.

20 ml of the following reaction solution was used in a process that theproline hydroxylase mutant with E27K amino acid residue mutation encodedby SEQ ID NO: 16 catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 10 wt/8 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. At 40 hours, a conversion rate was 94.53%, anddiastereomeric excess of the (2S,5S)-5-hydroxypiperidine-2-carboxylicacid was 98.32%.

20 ml of the following reaction solution was used in a process that theproline hydroxylase mutant with N165H amino acid residue mutationencoded by SEQ ID NO: 122 catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 10 wt and 6.67 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 10 wt and 6.67 wt respectively, after 40 hours, theconversion rate was 100% and 90.41% respectively, and diastereomericexcess of the (2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 99.89%.

According to another more preferable single-site mutant, 20 ml of thefollowing reaction solution was used in a process that the prolinehydroxylase mutant with K123D amino acid residue mutation encoded by SEQID NO: 104 catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 8 wt/5 wt/4 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 8 wt, 5 wt and 4 wt respectively, after 40 hours, theconversion rate was 100%, 97.83% and 89.09% respectively, anddiastereomeric excess of the (2S,5S)-5-hydroxypiperidine-2-carboxylicacid was 98.68%.

According to a multi-site mutant, 20 ml of the following reactionsolution was used in a process that the proline hydroxylase mutant withS37C+I223V amino acid residue mutation encoded by SEQ ID NO: 180catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 8 wt/5 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 8 wt and 5 wt respectively, after 40 h, the conversionrate was 100% and 93.99% respectively, and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 98.25%.

According to the multi-site mutant, 20 ml of the following reactionsolution was used in a process that the proline hydroxylase mutant with139R+Y35W amino acid residue mutation encoded by SEQ ID NO: 178catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 5 wt/3 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 5 wt and 3 wt respectively, after 40 h, the conversionrate was 100% and 94.45% respectively, and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 75.02%. Although theselectivity of the mutant is poorer, the catalytic activity thereof isapparently higher than that of the wild type proline hydroxylase.

According to a more preferable combined mutant, 20 ml of the followingreaction solution was used in a process that the proline hydroxylasemutant with I39R+K123D amino acid residue mutation encoded by SEQ ID NO:140 catalyzed L-piperidine acid to prepare the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of the reactionsolution comprised: 50 g/L of L-piperidine acid, 4 wt/3 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 4 wt and 3 wt respectively, after 40 h, the conversionrate was 97.35% and 92.08% respectively, and diastereomeric excess ofthe (2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 99.26%.

According to another more preferable combined mutant, 20 ml of thefollowing reaction solution was used in a process that the prolinehydroxylase mutant with S37C+I39K+I223V+K123D amino acid residuemutation encoded by SEQ ID NO: 160 catalyzed L-piperidine acid toprepare the (2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 mL of thereaction solution comprised: 50 g/L of L-piperidine acid, 3 wt/2 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. In the reaction system of which the amount ofrecombinase was 3 wt and 2 wt respectively, after 40 h, the conversionrate was 95.23% and 88.41% respectively, and diastereomeric excess ofthe (2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 99.90%.

Embodiment 6

An amino acid sequence PH1 (as shown in SEQ ID No:289 in the sequencelisting) independently designed and constructed by the inventor has 78%of homology with SEQ ID NO:2. The protein was used for catalyzing theL-piperidine acid to prepare (2S,5S)-5-hydroxypiperidine-2-carboxylicacid. 20 ml of the following reaction solution was used. 20 mL of thereaction solution comprised: 50 g/L of L-piperidine acid, 9 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. After 40 h, a conversion rate was 98.56% respectively,and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 98.45%.

Embodiment 7

1) A I39K+S33N amino acid residues mutation in SEQ ID NO:2 has 99% ofhomology with SEQ ID NO:2, and the amino acid sequence thereof is shownin SEQ ID NO: 290 in a sequence listing. Mutated enzyme encoded by thesequence was used for catalyzing the L-piperidine acid to prepare(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 ml of the followingreaction solution was used. 20 mL of the reaction solution comprised: 50g/L of L-piperidine acid, 8 wt/6 wt of recombinase, 62.2 g/L ofα-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5 mM of ammonium ferroussulfate, the reaction pH was 6.5, the reaction temperature was 10 DEGC., and the reaction time was 40 hours. At the end of the reaction, 100μL of the reaction system was taken, and then 200 μL of acetonitrile wasadded thereof, and 3000 μL of purified water was added after uniformlymixing, and the supernatant was collected by centrifugation at 10000 rpmfor 5 min for HPLC to determine the conversion rate. In the reactionsystem of which the amount of recombinase was 8 wt and 6 wtrespectively, after 40 hours, the conversion rate was 100% and 91.62%respectively, and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 98.89%.

2) A E27K+I39K+F28L+S31A amino acid residues mutation in the SEQ ID NO:2has 98.6% of homology with SEQ ID NO:2, and the amino acid sequencethereof is as shown in SEQ ID NO: 291 in the sequence listing. Themutated enzyme encoded by the sequence was used for catalyzing theL-piperidine acid to prepare (2S,5S)-5-hydroxypiperidine-2-carboxylicacid. 20 ml of the following reaction solution was used. 20 mL of thereaction solution comprised: 50 g/L of L-piperidine acid, 5 wt ofrecombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5mM of ammonium ferrous sulfate, the reaction pH was 6.5, the reactiontemperature was 10 DEG C., and the reaction time was 40 hours. At theend of the reaction, 100 μL of the reaction system was taken, and then200 μL of acetonitrile was added thereof, and 3000 μL of purified waterwas added after uniformly mixing, and the supernatant was collected bycentrifugation at 10000 rpm for 5 min for HPLC to determine theconversion rate. At 40 hours, the conversion rate was 98.75%respectively, and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 98.30%.

3) Base on the SEQ ID NO:289 sequence which has 78% of homology with theSEQ ID NO:2 and constructed in the embodiment 6, the mutation isperformed, after the E27K amino acid residue of the sequence wasmutated, the amino acid sequence as shown in SEQ ID NO: 292 in thesequence listing was obtained, the amino acid sequence has 97.8% of thehomology with the SEQ ID NO:2. The mutated proline hydroxylase encodedby the sequence was used for catalyzing the L-piperidine acid to prepare(2S,5S)-5-hydroxypiperidine-2-carboxylic acid. 20 ml of the followingreaction solution was used. 20 mL of the reaction solution comprised: 50g/L of L-piperidine acid, 7 wt of recombinase, 62.2 g/L ofα-ketoglutarate, 10.2 g/L of L-ascorbic acid, 5 mM of ammonium ferroussulfate, the reaction pH was 6.5, the reaction temperature was 10 DEGC., and the reaction time was 40 hours. At the end of the reaction, 100μL of the reaction system was taken, and then 200 μL of acetonitrile wasadded thereof, and 3000 μL of purified water was added after uniformlymixing, and the supernatant was collected by centrifugation at 10000 rpmfor 5 min for HPLC to determine the conversion rate. At 40 hours, theconversion rate was 97.25%, and diastereomeric excess of the(2S,5S)-5-hydroxypiperidine-2-carboxylic acid was 98.40%.

Control Example 1

In a process of the prior art for preparing(2S,5S)-5-hydroxypiperidine-2-carboxylic acid with a recombined prolinehydroxylase derived from Sinorhizobium mehloti, the highestdiastereomeric excess of the (2S,5S)-5-hydroxypiperidine-2-carboxylicacid was 90.7%, and 9.3% of a positional isomer(2S,3R)-3-hydroxypiperidine-2-carboxylic acid was generated(W02013169725A2).

Compared with the embodiment 5, the diastereomeric excess of hydroxylaseof SEQ ID NO:2 is 98.36%, and the diastereomeric excess of the controlexample 1 is 90.7%. It is clear that the selectivity of the hydroxylaseof SEQ ID NO:2 in the application is superior to the prior art.

Embodiment 8

Application of hydroxylases in Table 1 and Table 2 for preparingcis-4-hydroxy-L-proline, a reaction process thereof was as shown in FIG.2 .

A proline hydroxylase mutant mutated at K123D amino acid residue encodedby SEQ ID NO: 104 catalyzed L-piperidine acid to prepare thecis-4-hydroxy-L-proline. 20 ml of the following reaction solution wasused. 20 mL of the reaction solution comprised: 50 g/L of L-piperidineacid, 5 wt of recombinase, 62.2 g/L of α-ketoglutarate, 10.2 g/L ofL-ascorbic acid, 5 mM of ammonium ferrous sulfate, the reaction pH was6.5, the reaction temperature was 10 DEG C., and the reaction time was40 hours. At the end of the reaction, 100 μL of the reaction system wastaken, and then 200 μL of acetonitrile was added thereof, and 3000 μL ofpurified water was added after uniformly mixing, and the supernatant wascollected by centrifugation at 10000 rpm for 5 min for HPLC to determinethe conversion rate. At 40 hours, a conversion rate was 92.36%, anddiastereomeric excess was 99.30%.

The proline hydroxylase mutant mutated at S37C+I39K+I223V+K123D aminoacid residues encoded by SEQ ID NO: 160 catalyzed L-piperidine acid toprepare the cis-4-hydroxy-L-proline. 20 ml of the following reactionsolution was used. 20 mL of the reaction solution comprised: 50 g/L ofL-piperidine acid, 4 wt of recombinase, 62.2 g/L of α-ketoglutarate,10.2 g/L of L-ascorbic acid, 5 mM of ammonium ferrous sulfate, thereaction pH was 6.5, the reaction temperature was 10 DEG C., and thereaction time was 40 hours. At the end of the reaction, 100 μL of thereaction system was taken, and then 200 μL of acetonitrile was addedthereof, and 3000 μL of purified water was added after uniformly mixing,and the supernatant was collected by centrifugation at 10000 rpm for 5min for HPLC to determine the conversion rate. At 40 hours, theconversion rate was 99.47%, and the diastereomeric excess was 99.56%.

It is clear that many mutant hydroxylases in Table 1 and Table 2 may bealso applied to prepare cis-4-hydroxy-L-proline, and thecis-4-hydroxy-L-proline with high purity and high selectivity may beobtained too.

It is observed from the above description that the above embodiments ofthe application achieve the following technical effects: through usingthe SEQ ID NO:2 as a base sequence for screening mutated prolinehydroxylases, and by means of genetic engineering, multiple hydroxylaseswith remarkably improved catalytic activity and selectivity wereobtained. These hydroxylases have the characteristics of enabling asubstrate conversion rate to be high and catalyzing less specificpositional isomers, and are capable of specifically catalyzinghydroxylation of proline derivatives, especially, catalyzing thehydroxylation of L-piperidine acid to generate(2S,5S)-5-hydroxypiperidine-2-carboxylic acid (or named ascis-5-hydroxypiperidine acid) and catalyzing the hydroxylation ofL-proline to generate cis-4-hydroxy-L-proline.

The above are only the preferable embodiments of the application, butnot intended to limit the application. It is to be understood by thoseskilled in the art that the application may have various modificationsand changes. Any modifications, equivalent replacements, improvementsand the like made within the spirit and principles of the applicationshall fall within the scope of protection of the present application.

What is claimed is:
 1. A proline hydroxylase, comprising a proteinhaving the amino acid sequence of SEQ ID NO: 2 with the exception of amutation of one or more amino acids, wherein the mutation of one or moreamino acids must comprise E27K, and the mutation of one or more aminoacids is selected from the group consisting of: H14R, L16N, T25R, F26L,D30S, S33N, E34N, E34G, E34L, E34S, E34D, Y35W, Y35K, S37W, S37F, S37E,S37N, S37T, S37C, W40F, K41E, D54G, H55Q, S57L, I58T, I58Y, I58A, I58R,I58V, I58S, I58C, K86P, T91A, F95Y, C97Y, I98V, K106V, K106T, K106Q,F111S, K112E, K112R, S154A, K162E, L166M, I118F, I118V, I118R, H119R,H119F, I120V, K123D, K123N, K123Q, K123S, K123I, K123T, T130N, D134G,V135K, N165H, D173G, K209R, I223V and S225A and having prolinehydroxylase activity.
 2. The proline hydroxylase of claim 1, wherein themutation comprises any one of the combinations selected from the groupconsisting of: E27K+Y35W/K, E27K+K123/D/I/Q/S, E27K+N165H,E27K+S37C/E/F/N/W/T+I223V K123D/I/Q/S, I223V+S37C/E/F/N/W/T+E27K+N165H,wherein ‘/’ represents ‘or’.
 3. A method for producing anL-hydroxyproline derivative, wherein the method comprises: contacting anL-proline derivative substrate with the proline hydroxylase of claim 1to catalyze hydroxylation of the L-proline derivative substrate, toobtain the L-hydroxyproline derivative as shown in general formula (I):

wherein R₁ is selected from C₁-C₅ alkylene or C₂-C₅ alkenylene; R₂ isselected from C₀-C₄ alkylene or C₂-C₄ alkenylene; R₃ is selected fromhydroxyl, amino, C₁-C₆ alkoxy, aryloxy, C₁-C₆ alkyl sulfenyl or C₁-C₆aryl sulfenyl; and R₄ is selected from hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl or C₂-C₆ alkynyl.
 4. The method of claim 3, wherein the methodfurther comprises: adding

-oxoglutarate and O₂ as a common substrate and adding iron ions as acofactor.
 5. The method of claim 3, wherein the L-hydroxyprolinederivative is cis-4-hydroxy-L-proline or(2S,5S)-5-hydroxypiperidine-2-carboxylic acid.
 6. The method of claim 3,wherein the proline hydroxylase catalyzes hydroxylation of the L-prolinederivative substrate in a temperature of 5-45° C. to obtain theL-hydroxyproline derivative as shown in the general formula (I).
 7. Themethod of claim 6, wherein the proline hydroxylase catalyzeshydroxylation of the L-proline derivative substrate in a temperature of5-15° C. to obtain the L-hydroxyproline derivative as shown in thegeneral formula (I).